Homology-dependent gene silencing in plants and fungi: a number of variations on the same theme

Homology-dependent gene silencing is a phenomenon that occurs in a broad range of organisms and has implications for both basic and applied science. Gene silencing is a mechanism that controls invading transposons and provides protection against virus infections. It also has evolutionary implications in genome maintenance. Recent studies have begun to unravel the molecular mechanisms of this puzzling phenomenon.     

Composition and synthesis during G1 and S phase of a high mobility group-E/G component from Chinese hamster ovary cells

A perchloric acid soluble protein from the sedimented chromatin of blended Chinese hamster ovary (line CHO) cells has been isolated by guanidine hydrochloride gradient chromatography on Bio·Rex-70^® ion exchange resin. The amino acid composition of the protein (designated as CHO HMG-E/G) is similar to that of mouse HMG-E, but it differs from that of bovine HMG-14 and HMG-17 or any possible mixture of the two. CHO HMG-E/G incorporates [³²P]phosphate like HMG-14 and HMG-17 class proteins from other species, but all resolvable molecular species incorporate phosphate, and the more highly-phosphorylated band migrates faster, rather than slower, than the other in acid-urea gel systems. Incorporation of [³H]lysine into HMG-E/G following release from isoleucine deprivation G₁ block indicates that the protein is extensively synthesized during both the G₁ and S phases of the cell cycle.     

Deubiquitinating enzyme regulation of the p53 pathway: A lesson from Otub1

Deubiquitination has emerged as an important mechanism of p53 regulation. A number of deubiquitinating enzymes(DUBs) from the ubiquitin-specific protease family have been shown to regulate the p53-MDM2-MDMX networks. We recently reported that Otub1, a DUB from the OTU-domain containing protease family, is a novel p53 regulator. Interestingly, Otub1 abrogates p53 ubiquitination and stabilizes and activates p53 in cells independently of its deubiquitinating enzyme activity. Instead, it does so by inhibiting the MDM2 cognate ubiquitin-conjugating enzyme(E2) UbcH5. Otub1 also regulates other biological signaling through this non-canonical mechanism, suppression of E2, including the inhibition of DNA-damage-induced chromatin ubiquitination. Thus, Otub1 evolves as a unique DUB that mainly suppresses E2 to regulate substrates. Here we review the current progress made towards the understanding of the complex regulation of the p53 tumor suppressor pathway by DUBs, the biological function of Otub1 including its positive regulation of p53, and the mechanistic insights into how Otub1 suppresses E2.     

How to become neural crest: from segregation to delamination

The development of the neural crest up to the stage where they leave the neural tube can be observed as a series of concatenated but independent events that involve dorsalization of the neural plate/neural tube, neural crest induction, segregation and stabilization, epithelial to mesenchymal transition and delamination. During all these processes, the nascent neural crest cells are subjected to the influence of different signals and have to overcome competition for cell fate and apoptotic signals. In addition, striking rostrocaudal differences unveil how the regulatory cascades are somehow different but still can lead to the production of bona fide neural crest cells.     

Therapeutic potential of dental pulp stem cells and their derivatives: Insights from basic research toward clinical applications

For more than 20 years, researchers have isolated and identified postnatal dental pulp stem cells (DPSCs) from different teeth, including natal teeth, exfoliated deciduous teeth, healthy teeth, and diseased teeth. Their mesenchymal stem cell (MSC)-like immunophenotypic characteristics, high proliferation rate, potential for multidirectional differentiation and biological features were demonstrated to be superior to those of bone marrow MSCs. In addition, several main application forms of DPSCs and their derivatives have been investigated, including stem cell injections, modified stem cells, stem cell sheets and stem cell spheroids. In vitro and in vivo administration of DPSCs and their derivatives exhibited beneficial effects in various disease models of different tissues and organs. Therefore, DPSCs and their derivatives are regarded as excellent candidates for stem cell-based tissue regeneration. In this review, we aim to provide an overview of the potential application of DPSCs and their derivatives in the field of regenerative medicine. We describe the similarities and differences of DPSCs isolated from donors of different ages and health conditions. The methodologies for therapeutic administration of DPSCs and their derivatives are introduced, including single injections and the transplantation of the cells with a support, as cell sheets, or as cell spheroids. We also summarize the underlying mechanisms of the regenerative potential of DPSCs.     

The mouse pubic symphysis as a remodeling system: morphometrical analysis of proliferation and cell death during pregnancy, partus and postpartum

Marked changes in mice pubic symphysis occur by the end of pregnancy. Tissue remodeling involves a dynamic balance between cell proliferation and programmed cell death as well as changes in the extracellular matrix components. Therefore, it is important to consider both of these cellular behaviors when investigating the mechanism that regulates interpubic tissue remodeling, growth during late pregnancy and partus ensuring involution during the postpartum period. Proliferating and programmed death cells were identified by immunohistochemistry (proliferating cell nuclear antigen and TUNEL detection, respectively) and the rates at which these processes occurred were determined by morphometric analysis. The results demonstrated that cellular proliferation was intense during the period of ligament formation, from D15 to D18, thereafter abruptly declining on D19. From parturition (D19) onwards, an ever-increasing decline in the cellular proliferation levels could be observed. The quantitative analyses of cellular death showed opposite results when compared to cellular proliferation. During early pregnancy the cycle of cellular renovation was clearly proliferative and during late mouse pregnancy the cycle was directed by programmed cellular death. Although the high levels of cellular death during postpartum involution could be shown by the TUNEL-positive cells, we were unable to observed picnotic nucleus at the light microscopy.     

Interaction of FIE,a Polycomb protein,with pRb: a possible mechanism regulating endosperm development

Inactivation of the Arabidopsis protein FERTILIZATION INDEPENDENT ENDOSPERM (FIE) induces division of the central cell of the embryo sac, leading to endosperm development in the absence of fertilization. The mechanism whereby FIE regulates this process is unknown. We postulated that activation of central cell division in fie mutant plants might involve the retinoblastoma protein (pRb), a cell cycle regulatory element. Pull-down and surface plasmon resonance assays demonstrated that FIE interacts in-vitro with the pRb homologues from Arabidopsis (AtRb), maize (ZmRb) and human (HuRb). The interaction of FIE with ZmRB and HuRb in the yeast two-hybrid system supports the possibility that a FIE-pRb interaction may occur also in planta. Mutational analysis showed that this interaction does not occur via the LxCxE motif of the FIE protein nor via the pocket B domain of pRb. These results suggest that FIE may inhibit premature division of the central cell of the embryo sac, at least partly, through interaction with pRb, and suppression of pRb-regulated genes.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by R. G. Herrmann     

Microtubule organizing centers in plant cells: localization of a 49 kDa protein that is immunologically cross-reactive to a 51 kDa protein from sea urchin centrosomes in synchronized tobacco BY-2 cells

Summary A 49 kDa protein in tobacco BY-2 cells has been found to be cross-reactive with antibodies raised against a 51 kDa protein that was isolated from sea urchin centrosomes and identified as a microtubule-organizing center (MTOC) in animal cells. Tracing the fate of the 49 kDa protein during progression of the cell cycle in highly synchronized tobacco BY-2 cells revealed that this protein was colocalized with plant microtubules (MTs): the location of the 49 kDa protein coincided with preprophase bands (PPBs), mitotic spindles and phragmoplasts. Furthermore, between the M and G₁ phases, the 49 kDa protein was observed in the perinuclear regions, in which the initials of MTs are organizing to form cortical MTs. At the G₁ phase the location of the 49 kDa protein in the cell cortex coincided with that of the cortical MTs. It appeared that the 49 kDa protein in the cell cortex was transported as granules from the perinuclear regions. Thus, it is highly probable that the 49 kDa protein, which reacts with antibodies against the 51 kDa protein in sea urchin centrosomes, plays the role of an MTOC in plant cells. Thus, the mechanisms for organizing MTs in higher organisms appear to share a common protein, even though the organization of MTs is superficially very different in plant and animal cells.Abbreviations DAPI 4,6-diamidino-2-phenyl indole - MT microtubule - MTOC microtubule-organizing center - PAGE polyacrylamide gel electrophoresis - PBS phosphate-buffered saline - PPB preprophase band - SDS sodium dodecylsulfate     

Benzobijuglone, a novel cytotoxic compound from Juglans mandshurica, induced apoptosis in HeLa cervical cancer cells

A new quinone compound, p-hydroxymethoxybenzobijuglone (HMBBJ), isolated from Juglans mandshurica by bioassay-guided fractionation, showed cytotoxic activity against HeLa cell line. Its chemical structure was determined by NMR and HREIMS spectra. In this paper, its ability to induce apoptosis in HeLa cells was studied for the first time. After treated with HMBBJ, the growth of HeLa cells was inhibited and cells displayed typical morphological apoptotic characteristics. Data from flow cytometry analysis showed that the HeLa cell cycle was arrested in the G2/M phase by HMBBJ, and the apoptotic rate of HeLa cells increased in a dose-dependent manner. Meanwhile, HMBBJ increased the expression of caspase-8, -3 and Bax, decreased the expression of Bcl-2, and lowered the ΔΨ_m. These findings reveal that HMBBJ could efficiently induce HeLa cells apoptosis through mitochondria dependent pathway and activation of the caspase cascade, and it may be a potential chemotherapeutic candidate for the treatment of cancer.     

The transition from differential equations to Boolean networks: a case study in simplifying a regulatory network model

Methods for modeling cellular regulatory networks as diverse as differential equations and Boolean networks co-exist, however, without much closer correspondence to each other. With the example system of the fission yeast cell cycle control network, we here discuss these two approaches with respect to each other. We find that a Boolean network model can be formulated as a specific coarse-grained limit of the more detailed differential equations model for this system. This demonstrates the mathematical foundation on which Boolean networks can be applied to biological regulatory networks in a controlled way.     

Preprophase bands of microtubules and the cell cycle: Kinetics and experimental uncoupling of their formation from the nuclear cycle in onion root-tip cells

We have studied the timing of preprophase band (PPB) development in the division cycle of onion (Allium cepa L.) root-tip cells by combinations of immunofluorescence microscopy of microtubules, microspectrophotometry of nuclear DNA, and autoradiography of [³H]thymidine incorporation during pulse-chase experiments. In normally grown onion root tips, every cell with a PPB had the G2 level of nuclear DNA. Some were in interphase, prior to chromatin condensation, and some had varying degrees of chromatin condensation, up to the stage of prophase at which the PPB-prophase spindle transition occurs. In addition, autoradiography showed that PPBs can be formed in cells which have just finished their S phase, and microspectrophotometry enabled us to detect a population of cells in G2 which had no PPBs, these presumably including cells which had left the division cycle. The effects of inhibitors of DNA synthesis showed that the formation of PPBs is not fully coupled to events of the nuclear cycle. Although the mitotic index decreased 6-10-fold to less than 0.5% when roots were kept in 20 g·ml^-1 aphidicolin for more than 8 h, the percentage of cells containing PPBs did not decrease in proportion: the number of cells in interphase with PPBs increased while the number in prophase decreased. Almost the same phenomena were observed in the presence of 100 g·ml^-1 5-aminouracil and 40 g·ml^-1 hydroxyurea. In controls, all cells with PPBs were in G2 or prophase, but in the presence of aphidicolin, 5-aminouracil or hydroxyurea, some of the interphase cells with PPBs were in the S phase or even in the G1 phase. We conclude that PPB formation normally occurs in G2 (in at least some cases very early in G2) and that this timing can be experimentally uncoupled from the timing of DNA duplication in the cell-division cycle. The result accords with other evidence indicating that the cytoplasmic events of cytokinesis are controlled in parallel to the nuclear cycle, rather than in an obligatorily coupled sequence.Abbreviations APC aphidicolin - 5-AU 5-aminouracil - DAPI 4, 6-diamidino-2phenylindole - HU hydroxyurea - MI mitotic index - MT microtubule - PMSF phenylmethyl-sulfonyl fluoride - PPB preprophase band - %PPB percentage of cells with PPBs     

Retardation of cell cycle progression in yeast cells recovering from DNA damage: A study at the single cell level

Summary Pedigree analyses of individual yeast cells recovering from DNA damage were performed and time intervals between morphological landmark events during the cell cycle (bud emergence and cell separation), were recorded for three generations. The associated nuclear behavior was monitored with the aid of DAPI staining. The following observations were made: (1) All agents tested (X-rays, MMS, EMS, MNNG, nitrous acid) delayed the first bud emergence after treatment, which indicates inhibition of the initiation of DNA replication. (2) Cells that survived X-irradiation progressed further through the cell cycle in a similar way to control cells. (3) Progress of chemically treated cells became extremely asynchronous because surviving cells stayed undivided for periods of varying length. (4) Prolongation of the time between bud emergence and cell separation was most pronounced for cells treated with the alkylating agents MMS and EMS. This is interpreted as retardation of ongoing DNA synthesis by persisting DNA adducts. (5) Cell cycle prolongation in the second and third generation after treatment was observed only with MMS treated cells. (6) In all experiments, individual cells of uniformly treated populations exhibited highly variable responses.Abbreviations DAPI 4,6-diamidino-2-phenyl-indole - EMS ethyl methanesulfonate - MMS methyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine     

Isolation,expansion and neural differentiation of stem cells from human plucked hair: a further step towards autologous nerve recovery

Stem cells from the adult hair follicle bulge can differentiate into neurons and glia, which is advantageous for the development of an autologous cell-based therapy for neurological diseases. Consequently, bulge stem cells from plucked hair may increase opportunities for personalized neuroregenerative therapy. Hairs were plucked from the scalps of healthy donors, and the bulges were cultured without prior tissue treatment. Shortly after outgrowth from the bulge, cellular protein expression was established immunohistochemically. The doubling time was calculated upon expansion, and the viability of expanded, cryopreserved cells was assessed after shear stress. The neuroglial differentiation potential was assessed from cryopreserved cells. Shortly after outgrowth, the cells were immunopositive for nestin, SLUG, AP-2α and SOX9, and negative for SOX10. Each bulge yielded approximately 1 × 10⁴ cells after three passages. Doubling time was 3.3 (±1.5) days. Cellular viability did not differ significantly from control cells after shear stress. The cells expressed class III β-tubulin (TUBB3) and synapsin-1 after 3 weeks of neuronal differentiation. Glial differentiation yielded KROX20- and MPZ-immunopositive cells after 2 weeks. We demonstrated that human hair follicle bulge-derived stem cells can be cultivated easily, expanded efficiently and kept frozen until needed. After cryopreservation, the cells were viable and displayed both neuronal and glial differentiation potential.     

Toxoplasma gondii within skeletal muscle cells: a critical interplay for food-borne parasite transmission

Toxoplasma gondii infects virtually any nucleated cell type of warm-blooded animals and humans including skeletal muscle cells (SkMCs). Infection of SkMCs by T. gondii, differentiation from the highly replicative tachyzoites to dormant bradyzoites and tissue cyst formation are crucial for parasite persistence in muscle tissue. These processes are also prerequisites for one of the major routes of transmission to humans via undercooked or cured meat products. Evidence obtained in vitro and in vivo indicates that SkMCs are indeed a preferred cell type for tissue cyst formation and long-term persistence of T. gondii. This raises intriguing questions about what makes SkMCs a suitable environment for parasite persistence and how the SkMC–T. gondii interaction is regulated. Recent data from our laboratory show that differentiation of SkMCs from myoblasts to syncytial myotubes, rather than the cell type itself, is critical for parasite growth, bradyzoite formation and tissue cyst maturation. Myotube formation is accompanied by a permanent withdrawal from the cell cycle, and the negative cell cycle regulator cell division autoantigen (CDA)-1 directly or indirectly promotes T. gondii stage conversion in SkMCs. Moreover, host cell cycle regulators are specifically modulated in mature myotubes, but not myoblasts, following infection. Myotubes also up-regulate the expression of various pro-inflammatory cytokines and chemokines after T. gondii infection and they respond to IFN-γ by exerting potent anti-parasitic activity. This highlights that mature myotubes are active participants rather than passive targets of the local immune response to T. gondii which may also govern the interaction between SkMCs and the parasite.     

Culture of osteogenic cells from human alveolar bone: a useful source of alkaline phosphatase

The aim of this study was to obtain membrane-bound alkaline phosphatase from osteoblastic-like cells of human alveolar bone. Cells were obtained by enzymatic digestion and maintained in primary culture in osteogenic medium until subconfluence. First passage cells were cultured in the same medium and at 7, 14, and 21 days, total protein content, collagen content, and alkaline phosphatase activity were evaluated. Bone-like nodule formation was evaluated at 21 days. Cells in primary culture at day 14 were washed with Tris-HCl buffer, and used to extract the membrane-bound alkaline phosphatase. Cells expressed osteoblastic phenotype. The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10.0. This enzyme also hydrolyzes ATP, ADP, fructose-1-phosphate, fructose-6-phosphate, pyrophosphate and beta-glycerophosphate. PNPPase activity was reduced by typical inhibitors of alkaline phosphatase. SDS-PAGE of membrane fraction showed a single band with activity of approximately 120 kDa that could be solubilized by phospholipase C or Polidocanol.     

Post-irradiation DNA synthesis inhibition and G2 phase delay in radiosensitive body cells from non-Hodgkin's lymphoma patients: an indication of cell cycle defects

In the present study, both post-irradiation DNA synthesis and G₁ phase accumulation were analyzed in lymphoblastoid cell lines (LCLs) and fibroblast cell strains derived from (Saudi) patients with non-Hodgkin's lymphoma (NHL), ataxia telangiectasia (AT), AT heterozygotes and normal subjects. A comparison of the percent DNA synthesis inhibition (assayed by ³H-thymidine uptake 30 min after irradiation), and a 24 h post-irradiation G₂ phase accumulation determined by flow cytometry placed the AT heterozygotes and the NHL patients in an intermediate position between the normal subjects (with maximum DNA synthesis inhibition and minimum G₂ phase accumulation) and the AT homozygotes (with minimum DNA synthesis inhibition and maximum G₂ accumulation). The similarity between AT heterozygotes and the NHL patients with respect to the two parameters studied after irradiation was statistically significant. The data indicating a moderate abnormality in the control of cell cycle progression after irradiation in the LCLs and fibroblasts from NHL patients may explain the enhanced cellular and chromosomal radiosensitivity in these patients reported by us earlier. In addition to demonstrating a link between cell cyle abnormality and radiosensitivity as a possible basis for cancer susceptibility, particularly in the NHL patients, the present studies emphasized the usefulness of the assay for 24 h post-irradiation G₂ phase accumulation developed by Lavin et al. (1992) in characterizing AT heterozygote-like cell cycle anomally in cancer patients irrespective of whether they carried the AT gene or any other affecting the cell cycle.     

Nucleosome-dependent escape of tumor cells from natural-killer-mediated lysis: nucleosomes are taken up by target cells and act at a postconjugation level

 Our previous data suggested that chromatin fragments released from dead cells into the extracellular medium could be involved in the impairment of natural-killer (NK)-mediated cytotoxicity reported in cancer patients. In the present study, an inhibition of the NK-mediated lysis was obtained in vitro by nucleosome addition to different tumor target cells, independently of their sensitivity to NK-mediated lysis. We observed a rapid endocytosis and degradation of nucleosomes by K562 tumor target cells and (although to a much lesser extent) a binding to a subpopulation of lymphocytes. Nucleosomes impaired neither the conjugation step nor the expression of adhesion molecules at the effector (CD11a, CD18, CD2) or target (CD54, CD58) cell surface. On the contrary, flow-cytometry analysis of the conjugation suggested that nucleosomes might stabilize the conjugates. Investigations of the killing process showed that nucleosomes decreased the NK cytotoxic potential without modifying Ca²⁺-dependent lethal-hit-delivery kinetics. The cytotoxic potential was not restored by increasing the available magnesium and calcium concentrations in the extracellular medium. Taken together, the results suggest that the inhibition of NK-mediated lysis by nucleosomes may result from alterations of the NK mechanism at the postconjugation level and after lethal-hit delivery. Hence, the inhibition could involve a delay in the recycling of effector cells, or a resistance of tumor target cells to NK cells. Received: 7 October 1996 / Accepted: 12 November 1996     

Antiproliferative and apoptotic potential of Glycyrrhizin against HPV16+ Caski cervical cancer cells: A plausible association with downreguation of HPV E6 and E7 oncogenes and Notch signaling pathway

Cervical cancer (CCa) is the second most frequent carcinoma in females and human papilloma virus (HPV) oncoproteins are regarded as one of the critical etiological agent. Despite recent advances in screening and management of CCa, still it remains the deadliest carcinoma as advanced and metastatic stages are mostly incurable. This urges for the development of newer therapeutic interventions. The current was aimed to investigate the antiproliferative and apoptotic potential of glycyrrhizin (Gly) against HPV16⁺ CaSki CCa cells. Our findings substantiated that Gly exerted antiproliferative effects on the CaSki cells by obstructing their proliferation rate. Gly substantially enhanced apoptosis in Caski cells in a dose-dependent manner via augmenting the generation of ROS, DNA fragmentation and disruption of the mitochondrial membrane potential. Gly mediated apoptosis in CaSki cells was found to be due to activation of caspase-8 and capsase-9 along with the modulation of pro-and anti-apoptotic gene expression. Moreover, Gly halts the progression of CaSki cells at G₀/G₁ phase which was found to be due to reduced expression of cyclin D1 and cyclin-dependent kinase 4 (CDK4) along with the enhanced expression of CDK inhibitor p21^Cip1. Further, Gly downregulates the expression of HPV oncoproteins (E6 & E7) along with the inhibition of Notch signaling pathway. Taken together, Gly represents as a potential therapeutic modality for CCa which could rapidly be translated for clinical studies.